An EGF receptor targeting Ranpirnase-diabody fusion protein mediates potent antitumour activity in vitro and in vivo

Cancer Letters 357 (2015) 364–373

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

An EGF receptor targeting Ranpirnase-diabody fusion protein mediates potent antitumour activity in vitro and in vivo Stefan Kiesgen a, Michaela A.E. Arndt a,b, Christoph Körber c, Ulrich Arnold d, Tobias Weber a, Niels Halama a, Armin Keller a, Benedikt Bötticher a, Anne Schlegelmilch a, Nora Liebers a, Martin Cremer a, Christel Herold-Mende e,f, Gerhard Dyckhoff f, Philippe A. Federspil g, Alexandra D. Jensen h,i, Dirk Jäger a, Roland E. Kontermann j, Walter Mier k, Jürgen Krauss a,* a Department of Medical Oncology, National Center for Tumor Diseases, Heidelberg University Hospital, Im Neuenheimer Feld 460, Heidelberg 69120, Germany b Immunotherapy Program, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg 69120, Germany c Institute of Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, Heidelberg 69120, Germany d Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle 06120, Germany e Division of Experimental Neurosurgery, Department of Neurosurgery, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany f Molecular Cell Biology Group, ENT Department, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany g Department of Otorhinolaryngology, Head and Neck Surgery, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany h Department of Radiation Oncology, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany i Heidelberg Ion Therapy Center (HIT), Im Neuenheimer Feld 450, Heidelberg 69120, Germany j Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, Stuttgart 70569, Germany k Department of Nuclear Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany

A R T I C L E

I N F O

Article history: Received 27 August 2014 Received in revised form 19 November 2014 Accepted 20 November 2014 Keywords: immunoRNase Diabody EGFR Ranpirnase Onconase

A B S T R A C T

Cytotoxic ribonucleases such as the leopard frog derivative Ranpirnase (Onconase®) have emerged as a valuable new class of cancer therapeutics. Clinical trials employing single agent Ranpirnase in cancer patients have demonstrated significant clinical activity and surprisingly low immunogenicity. However, dose-limiting toxicity due to unspecific uptake of the RNase into non-cancerous cells is reached at relatively low concentrations of > 1 mg/m2. We have in the present study generated a dimeric antiEGFR Ranpirnase-diabody fusion protein capable to deliver two Ranpirnase moieties per molecule to EGFR-positive tumour cells. We show that this compound mediated far superior efficacy for killing EGFR-positive tumour cells than a monomeric counterpart. Most importantly, cell killing was restricted to EGFR-positive target cells and no dose-limiting toxicity of Ranpirnase-diabody was observed in mice. These data indicate that by targeted delivery of Ranpirnase non-selective toxicity can be abolished and suggests Ranpirnase-diabody as a promising new drug for therapeutic interventions in EGFR-positive cancers. © 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction The epidermal growth factor receptor (EGFR) belongs to the ErbB/ HER family of transmembrane receptors involved in modulation of cell proliferation, survival, migration, and differentiation [1]. Mutations clustering in functionally relevant hotspots of the EGF receptor commonly lead to enhanced intrinsic tyrosine kinase activity or dysregulation of suppressive domains and contribute to the pathogenesis and progression of epithelial cancers. Moreover, increased EGFR expression has been

* Corresponding author. Tel.: +49 6221 565922; fax: +49 6221 565317. E-mail address: [email protected] (J. Krauss). http://dx.doi.org/10.1016/j.canlet.2014.11.054 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.

linked to an adverse prognosis in patients with head and neck, bladder, ovarian and cervical cancers [2]. Consequently, inhibition of EGFR function is considered to be a promising therapeutic approach. Both small molecule tyrosine kinase inhibitors (TKIs) as well as monoclonal antibodies (mAbs) with specificity for the extracellular ligand-binding domain have been approved by the FDA as EGFR-targeted cancer therapies. However, EGFR-targeted cancers frequently develop resistance mechanisms towards these compounds, rendering them ineffective over time [3–5]. For example, although TKIs have been shown to impressively prolong progression-free survival of non-small cell lung cancer patients harbouring gain-of-function EGFR mutants [6–8], almost all of these patients eventually relapsed due to acquired resistance to antiEGFR treatment [3,9]. Antineoplastic effects of approved EGFR-targeting mAbs are mediated by cell signalling interference, recruitment of

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immune effector cells (antibody-dependent cell-mediated cytotoxicity, ADCC) and activation of the complement system (complementdependent cytotoxicity, CDC). Since EGFR is rapidly internalised upon binding to several mAbs [10], the receptor is well suited for the targeted delivery of cytotoxic compounds into cytosolic compartments. Ribonucleases have been proposed as a valuable payload for the targeted delivery by monoclonal antibodies and antibody fragment derivatives [11]. To trigger cytotoxicity, immunoRNases need to be efficiently routed to cytosolic substrates and evade inhibition by the species-specific RNase inhibitor protein [12]. Amphibian Ranpirnase (Onconase®) has gained attention as an anti-tumoural agent since it is not inactivated by the human RNase inhibitor and induces apoptosis via degradation of cytosolic tRNA substrates and microRNAs being differentially expressed in cancerous tissues [13–16]. Furthermore, activation of caspases and upregulation of pro-apoptotic proteins have been reported as mechanisms of action [17,18]. Despite its amphibian origin, surprisingly little immunogenicity has been reported in clinical trials [19,20]. Thus, Ranpirnase appears to be an attractive effector compound for antibody-based delivery to malignant cells. We have previously shown that dimerisation of monomeric RNase-scFv fusion proteins carrying either human Angiogenin or the Ranpirnase homolog Rana pipiens liver RNase I (rapLRI) as effector moiety markedly enhanced cytotoxicity against CD22 + lymphoma cells in vitro [21,22]. Here we report on the generation of a new dimeric immunoRNase composed of Ranpirnase being fused to a humanised diabody fragment with the specificity of Cetuximab. We show here that this dimeric immunoRNase is several orders of magnitude more cytotoxic towards EGFR-expressing tumour cells than its monomeric counterpart and exhibits significant antitumour activity in a murine A431 xenograft model. Moreover, off-target cytotoxicity of Ranpirnase is completely abolished in the fusion protein configuration. Materials and methods Cell lines and proteins Human head and neck squamous cell carcinoma (HNSCC) cell lines HNO97 (oral cavity), HNO211 (oropharynx), HNO410 (hypopharyngeal lymph node metastasis) and HNO210 (larynx) were established from surgical specimens of HNSCC patients after informed consent and approval by the ethics committee of the Faculty of Medicine, Heidelberg University [23]. The patient-derived primary HNSCC cell lines and the commercial HNSCC cell lines FaDu (pharynx, ATCC HTB-43) and CAL27 (tongue, ATCC CRL-2095) as well as the human epidermoid carcinoma cell line A431 (ATCC CRL-1555) and human breast adenocarcinoma cell line MCF7 (ATCC HTB-22) were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, Taufkirchen, Germany) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich) in a humidified incubator with 5% CO2 at 37 °C. Human Burkitt’s lymphoma cell line Raji (ATCC CCL-86) was cultivated in RPMI1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin under the same conditions. The cell line HEK293-6E (licensed from the National Research Council, Biotechnological Research Institute, Montreal, Canada) was cultured in F17 medium (Invitrogen, Life Technologies, Darmstadt, Germany) supplemented with 0.1% Kolliphor P188 (Sigma-Aldrich), 4 mM glutamine (Invitrogen) and 25 μg/ml G418 (Carl Roth, Karlsruhe, Germany) in shaker incubators at 37 °C, 5% CO2 and 120 rpm. Cetuximab was commercially obtained from Merck Serono (Darmstadt, Germany). Recombinant Ranpirnase was kindly provided by Kuslima Shogen, Alfacell Corporation, USA.

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For generation of the Ranpirnase-scFv and Ranpirnase-diabody genes, the DNA encoding Ranpirnase-(G4S)3 was modified by PCR to introduce flanking AgeI and BamHI restriction sites. DNA encoding the humanised anti-EGFR scFv and the humanised anti-EGFR diabody with BamHI restriction site at the 5′-end of the variable heavy chain gene and NotI restriction site at the 3′-end of the variable light chain gene were generated by PCR followed by ligation together with the AgeI/BamHI-digested Ranpirnase-(G4S)3 gene fragment into the AgeI/NotI-linearised mammalian expression vector pSECTagA (Invitrogen).

Expression and purification of scFv, diabody, Ranpirnase-scFv and Ranpirnase-diabody Soluble expression of anti-EGFR scFv and anti-EGFR diabody in the periplasmatic space of Escherichia coli TG1 (Stratagene, Agilent Technologies, Santa Clara, CA, USA) was performed as described previously [27]. The periplasmic extract containing scFv or diabody was thoroughly dialysed against SP10 buffer (20 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) at 4 °C and purified by immobilised metal ion affinity chromatography (IMAC) using SP-10 equilibrated Ni-NTA columns (GE Healthcare, Muenchen, Germany). Bound proteins were eluted in SP-buffer containing increasing imidazole concentrations (10–500 mM). Ranpirnase-scFv and Ranpirnase-diabody were transiently expressed in HEK2936E cells in shaking flasks (Falcon, Becton Dickinson, Heidelberg, Germany). Endofree plasmid DNA (1 μg/ml final culture volume) and polyethylenimine (2 μg/ml final culture volume; Polysciences, Warrington, PA, USA) were prepared separately in 1/20 of final culture volume in F17 medium without G418, mixed, incubated at room temperature for 3 min and added to HEK293-6E cells at a density of 1.7–2 × 106 cells/ ml. One day after transfection, cells were supplemented with 0.5% (w/v) tryptone TN1 (Organotechnie S.A.S, La Courneuve, France). ImmunoRNase-containing supernatant was harvested five days after transfection, dialysed against SP20 buffer (20 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and concentrated using a SARTOFLOW® Slice 200 benchtop crossflow system (Sartorius, Goettingen, Germany). Following IMAC purification using a multi-step imidazole gradient, elution fractions containing recombinant protein were pooled and dialysed against PBS at 4 °C. All proteins were further purified to homogeneity by size exclusion chromatography using either a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) for scFv and Ranpirnase-scFv, respectively, or a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) for diabody and Ranpirnase-diabody, respectively, on an ÄKTA FPLC system (GE Healthcare). Purified antibody fragments and immunoRNases were analysed by SDS-PAGE under reducing conditions and stained with Simply Blue Safe Stain (Invitrogen) or by Western blot, using the anti-(c-myc)-peroxidase-conjugated mAb 9E10 (Roche, Penzberg, Germany) followed by ECL Plus (ThermoScientific, Rockford, IL, USA) detection. Purity and correct size of recombinant proteins were monitored under native conditions on a Superdex 200 10/300 GL column (GE Healthcare). The column was calibrated using gel filtration molecular weight standards (GE Healthcare, 17-0442-01 and 28-4038-42). Elution profiles were recorded by monitoring the absorbance at 280 nm and elution volumes were determined with the Unicorn software 5.11.

Cell surface binding measurements Equilibrium binding curves were determined by incubating A431 cells with serial dilutions of either scFv, diabody, Ranpirnase-scFv, Ranpirnase-diabody or Cetuximab. Bound antibody fragment or immunoRNase was detected by anti-c-myc mAb 9E10 (Roche) and fluorescein isothiocyanate (FITC)-labelled goat-anti-mouse IgG (Jackson ImmunoResearch, Suffolk, UK). Bound chimeric mAb Cetuximab was detected by FITClabelled rabbit-anti-human IgG (Jackson ImmunoResearch). Fluorescence of stained cells was measured on a FACS Canto II flow cytometer (Becton Dickinson) using the FACS Diva Software (Becton Dickinson). The background fluorescence was subtracted from measured median fluorescence and relative affinities were calculated by nonlinear regression using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). EGFR cell surface expression was analysed by flow cytometry using Cetuximab at saturating concentrations (10 μg/ml). Bound antibody was detected as described above. To determine relative EGFR expression median fluorescence signals obtained with Cetuximab were corrected for the cell line-specific median fluorescence intensities from control stainings (isotype control). All measurements were performed in triplicates.

Cloning of scFv, diabody, Ranpirnase-scFv and Ranpirnase-diabody

Determination of ribonucleolytic activity

The bacterial expression vector pAB1 [24] encoding the humanised anti-EGFR scFv IZI08 [25] derived from the C225 antibody [26] was used as template for constructing the anti-EGFR diabody gene. For this purpose, the linker DNA sequence between the VH and VL gene segments was shortened to 15 nucleotides encoding for amino acids GGGGS by means of splicing by overlap extension polymerase chain reaction (SOE-PCR). The resulting diabody gene segment was digested with NcoI and NotI and cloned into bacterial expression vector pAB1. The gene of Ranpirnase (UniProtKB/Swiss-Prot: P22069.2) with an additional gene segment coding for a C-terminal (G4S)3 linker was synthesised for optimised expression in mammalian cells (Entelechon, Bad Abbach, Germany).

Ribonucleolytic activity of Ranpirnase and Ranpirnase fusion proteins was measured using the fluorogenic substrate 6-Carboxyfluorescein-dArUdGdA-BlackHole-Quencher-1 (6-FAM-dArUdGdA-BHQ-1) (http://www.biomers.net/, Ulm, Germany). Reactions were performed in black 96-well plates in 100 mM MESNaOH buffer (pH 6.0) containing 100 mM NaCl and 6-FAM-dArUdGdA-BHQ-1 (5 nM) at (25 ± 2) °C in a total reaction volume of 200 μl per well. Fluorescence increase after cleavage of substrate was monitored over time using an Infinite F200Pro microplate reader (Tecan, Maennedorf, Switzerland) with a 485/535 nm (excitation/emission) filter set. Buffer was used as negative control and an excess concentration of RNase A served as positive control. Values of kcat/KM were calculated using the equation:

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kcat K M =

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(ΔF Δt ) (Fmax − F0 ) ⋅ [E]

where ΔF/Δt represents the initial reaction velocity, F0 the initial fluorescence intensity before addition of RNase, Fmax the fluorescence intensity after complete cleavage of the substrate by excess RNase A and [E] the RNase concentration. At least three independent assays with each assay containing triplicates were performed.

Internalisation For internalisation studies, Ranpirnase, Ranpirnase-scFv, Ranpirnase-diabody and Cetuximab were fluorescence-labelled by incubating the corresponding protein solutions in PBS with a 5 to 15-fold molar excess of Alexa 647 N-hydroxysuccinimide ester (Invitrogen) for 60 min at room temperature. Unbound dye was removed by extensive dialysis against PBS. A431 cells were grown on coverslips in 24-well plates at 37 °C and 5% CO2 for 24 h. Labelled Ranpirnase, Ranpirnase-scFv, Ranpirnasediabody and Cetuximab were added for 5, 10, 15, 30, 60, 120 and 240 min at 100 nM. Additional time points (12 and 24 h) were investigated for Ranpirnase. Noninternalised, surface-bound protein was removed by incubation with stripping buffer (100 mM Glycin, 100 mM NaCl, pH 2.5) for 5 min on ice. Cells were washed twice with PBS and fixed with 4% (w/v) paraformaldehyde dissolved in PBS for 10– 20 min at room temperature. Nuclei of cells were stained with DAPI (2.5 μg/ml, Sigma). Uptake of fluorescence-labelled proteins was analysed using a Leica TCS SP5 microscope equipped with a 63x HCX PL APO (1.45 NA) objective. Confocal images of 9–10 cells per time point were acquired at constant laser and PMT settings. Uptake was measured as mean fluorescence intensity per cell using ImageJ software (http://imagej.nih.gov/ij/). Results obtained from individual cells were normalised to the average mean fluorescence intensity of all cells analysed at the latest time point examined.

In vitro cytotoxicity assay In order to determine antitumour activity of recombinant proteins in vitro, cells were seeded in 96-well plates (Falcon) and incubated with different concentrations of protein or buffer in a total volume of 110 μl at 37 °C, 5% CO2 for 72 h. Cell viability of adherent cell lines (A431, HNO97, HNO211, HNO410, HNO210, CAL27, FaDu, MCF7) was determined by addition of 20 μl of a 5 mg/ml solution of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) diluted in PBS. After incubation at 37 °C, 5% CO2 for 4 h, the medium was replaced with 100 μl solubilisation solution (10% SDS (w/v) and 0.6% acetic acid (v/v) in dimethyl sulfoxide) per well. Plates were incubated for 5 min at room temperature followed by 5 min gentle agitation to allow solubilisation of formazans. Viability of the suspension cell line Raji was analysed by adding 10 μl alamarBlue® (ThermoScientific) per well followed by incubation at 37 °C, 5% CO2 for 4 h. Absorbance of both MTT-treated and alamarBlue®treated cells was measured at 570 nm (reference: 620 nm) using an Infinite F200Pro microplate reader (Tecan). Viability of treated cells was expressed relative to buffertreated control cells. The concentration of recombinant proteins required to inhibit cell viability by 50% relative to buffer-treated control cells (IC50) was calculated using GraphPad Prism 5.0. Each assay was perfomed in triplicates.

Subcutaneous xenograft tumour model Tumours were established by inoculating 5 × 106 A431 cells in a total volume of 100 μl PBS subcutaneously into the right flank of 6 to 8 weeks old female BALB/c nude mice (Janvier, France). When tumours were palpable (~25 mm3) mice were randomised into four groups and treated with 100 μl of either 60 μg Ranpirnasediabody (n = 8), 40 μg diabody (n = 2), 20 μg Ranpirnase (n = 2) or buffer-control (PBS, n = 9) by intraperitoneal injection for five consecutive days. Mice were examined daily, tumour size and mouse weight was measured every 2–3 days. Tumour diameters were measured with a digital caliper and tumour volume was calculated using the formula (length) × (width)2 × 0.5. Animals were sacrificed by cervical dislocation if tumour ulceration occurred or tumour volume reached 1000 mm3. All animal experiments were approved by the local Animal Care and Use Committee and carried out in accordance with the German animal protection law. Radioactive labelling Radioactive labelling using the radioactive isotope 125I for scintigraphic imaging or 131I for the biodistribution studies was performed by the Chloramine-T-Method. For this purpose, an aliquot of a solution of NaI (approximately 20 MBq of 125I or 10 MBq 131I, Perkin Elmer, Rodgau, Germany) was mixed with 50 μl PBS. Under stirring 70 μl of a solution of the protein to be iodinated (approximately 2 mg/ml) in PBS and 10 μl of a solution of 5 μg Chloramine-T in PBS were added subsequently. After 30 sec, 50 μl of a saturated solution of methionine in PBS was added to quench the reaction. The reaction mixture was separated by size exclusion chromatography on a NAP-5 column (GE Healthcare) using 0.9% NaCl as the eluent. Biodistribution and scintigraphy For biodistribution and scintigraphy studies an activity amount of 0.5–10 MBq of the radiolabelled tracer was applied. The 131I-labelled tracer was applied via the tail vein. After 24 h and 48 h the animals were sacrificed and tumour as well as selected organs (heart, lung, spleen, liver, kidney, intestine and femur) dissected (n = 3 per group and time point). Samples of the tissues were blotted dry, weighed and counted in a gamma counter. The results obtained are expressed as percentage of the applied dose per gram of tissue [%ID/g]. Alternatively, the animals were studied by planar scintigraphy using the 125I-labelled immunoRNase. For this purpose the animals were anesthetised by inhalation narcosis, the tracer was injected via the tail vein and the distribution non-invasively followed in the scanner. Statistical analysis The Mann–Whitney test (one-tailed) was used to test the statistical significance between the area under the tumour volume curves of mice treated with Ranpirnase-diabody and mice treated with buffer control until day 14 after tumour transplantation, which allowed inclusion of data sets of all mice before any mice had to be taken out due to tumour progression or tumour ulceration. Survival data were analysed using log-rank analysis. A value of p < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software).

Results Caspase 3/7 activity Caspase 3/7 activity was measured using the Caspase Glo 3/7 assay (Promega, Mannheim, Germany) following the manufacturer’s instructions. Briefly, cells were seeded in white 96-well plates (Greiner, Frickenhausen, Germany) and incubated with 100 nM of protein or buffer control in a total volume of 100 μl at 37 °C, 5% CO2 for 24 h. A volume of 100 μl of caspase substrate was added per well and plates were gently mixed using a plate shaker at 500 rpm for 30 sec. After 2 h incubation at room temperature, luminescence was measured using an Infinite F200Pro microplate reader (Tecan). Background luminescence of the medium was subtracted from experimental values and luminescence of treated cells was related to luminescence of buffer control, corresponding to caspase 3/7 activity of treated cells in relation to basal caspase 3/7 activity. All experiments were performed in triplicates.

Phosphoprotein and total protein quantification Multiplex phosphoprotein and total protein quantification was performed on cultured cells as described previously [28,29]. To detect phosphoproteins and corresponding total proteins, lysates were prepared according to the manufacturer’s instructions (BioRad Laboratories, Hercules, CA, USA). A two-laser array reader simultaneously quantified all proteins of interest. Data analysis was performed using Bio-Plex Manager 4.1.1 (BioRad Laboratories, Hercules, CA, USA). In the analysis the ratios of phosphorylated protein to total protein were calculated and used to detect effects of specific intervention (i.e. Cetuximab, Ranpirnase-diabody). Analysis was performed as described previously [29].

Generation, expression and purification of antibody fragments and immunoRNases For the targeted delivery of Ranpirnase to EGFR-expressing tumour cells a humanised scFv [25] and derived diabody fragment with specificity of the clinically established mAb Cetuximab (Erbitux®) were employed as targeting moieties. The diabody fragment was generated by genetically shortening the linker between the VH and VL domain of the anti-EGFR scFv fragment from 15 ((G4S)3) to 5 amino acid residues (G 4 S). Subsequently, the Ranpirnase encoding gene was fused either to the scFv or diabody fragment via a flexible (G4S)3 linker yielding immunoRNases Ranpirnase-scFv or Ranpirnase-diabody, respectively (Fig. 1). Antibody fragments were produced as soluble proteins in the periplasm of bacteria whereas the two immunoRNases were produced into cell culture supernatants after transient transfection of mammalian cells. Recombinant proteins purified by IMAC followed by size exclusion chromatography yielded homogeneous protein preparations as demonstrated by analytical size exclusion chromatography (Fig. 2). All proteins eluted from a calibrated Superdex 200 column as single peaks at retention times correlating with the size of monomeric scFv (28 kDa),

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Fig. 1. Schematic presentation of immunoRNases. Ranpirnase (Ran) was either fused to an anti-EGFR scFv yielding the immunoRNase Ranpirnase-scFv or to an anti-EGFR diabody fragment yielding the immunoRNase Ranpirnase-diabody. VH: variable domain of the heavy chain, VL: variable domain of the light chain. A c-myc tag was used for detection and a His6 tag for purification. N-terminus (NH2) and C-terminus (COOH) are indicated.

monomeric immunoRNase (Ranpirnase-scFv, 41 kDa), non-covalently associated diabody fragment (55 kDa) and dimeric immunoRNase (Ranpirnase-diabody, 81 kDa). Production yields after final purification were 1.9 mg/l for the scFv, 2.4 mg/l for the diabody, 0.9 mg/l for Ranpirnase-scFv and 1.5 mg/l for Ranpirnase-diabody.

Functional characterisation of recombinant proteins Binding activities of scFv, diabody, Ranpirnase-scFv, Ranpirnasediabody and Cetuximab to cell surface-expressed EGFR were analysed by flow cytometry. Determination of apparent equilibrium dissociation constants (KD) was performed by flow cytometry using A431 cells (Table 1). The scFv fragment bound to the cells with a KD value of 9.6 nM whereas affinity was increased for the diabody fragment (KD = 6.5 nM) that well corresponds with the binding affinity constant of the parental mAb Cetuximab (KD = 6.8 nM). Binding affinity of the Ranpirnase-scFv fusion protein was slightly decreased (1.5-fold) when compared to the scFv fragment alone. Fusion of Ranpirnase to the diabody fragment (Ranpirnase-diabody), however, did not alter binding affinity when compared with the diabody fragment or Cetuximab. Ribonucleolytic activity of Ranpirnase and derived fusion proteins was measured by their ability to cleave the fluorogenic substrate 6-FAM-dArUdGdA-BHQ-1 (Table 1). Formation of pyroglutamate from the N-terminal glutamine of Ranpirnase is essential for its enzymatic activity [30]. Ranpirnase was therefore fused at the N-termini of the antibody fragments to not interfere with glutamine cyclisation. The presence of the antibody fragments, however, slightly affected the ribonucleolytic activity of Ranpirnase. Ranpirnase-scFv exhibited 1.3-fold decreased enzymatic activity when compared with Ranpirnase alone with k cat /K M values of 8.7 × 10 3 M −1 s −1 and 11.3 × 103 M−1 s−1, respectively. The catalytic activity of Ranpirnasediabody correlated very well with the number of Ranpirnase payloads per molecule with a catalytic activity twice the kcat/KM value of the monomeric immunoRNase (kcat/KM = 17.1 × 103 M−1 s−1). Besides binding affinity and enzymatic activity internalisation of an immunoRNase is an important step for the cytosolic delivery of the effector domain. We therefore monitored by confocal laser-scanning microscopy the internalisation of fluorescence-labelled immunoRNases and the current standard mAb Cetuximab into EGFR-expressing A431 cells over time (Fig. 3). Ranpirnase-scFv, Ranpirnase-diabody and

Table 1 Characterisation of immunoRNases for binding affinity and ribonucleolytic activity in comparison to respective antibodies or Ranpirnase.

Fig. 2. Analytical size exclusion chromatography of purified proteins. Proteins were purified to homogeneity and analysed under native conditions on a calibrated Superdex 200 column. (A) Diabody (55 kDa) and scFv (28 kDa) purified from the periplasm of E. coli TG1, respectively. (B) Ranpirnase-diabody (81 kDa) and RanpirnasescFv (41 kDa) purified from the supernatant of transiently transfected HEK29366E cells, respectively. Elution volumes are indicated.

scFv Diabody Cetuximab Ranpirnase-scFv Ranpirnase-diabody Ranpirnase

KD ± SE (nM)*

kcat/KM ± SE (103 M−1s−1)**

9.6 ± 1.2 6.5 ± 0.5 6.8 ± 0.7 14.7 ± 1.2 6.1 ± 0.6 n.a.

n.a. n.a. n.a. 8.7 ± 0.4 17.1 ± 1.7 11.3 ± 1.0

* Binding affinities (KD) on A431 cells were calculated from the equilibriumbinding curves as measured by flow cytometry. ** kcat/KM values for cleavage of 6-FAM-dArUdGdA-BHQ-1 were determined as described in materials and methods. SE, standard error; n.a., not applicable.

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its maximum after only 5 min incubation time (Fig. 3B). In contrast to the antibody constructs, the intrinsic internalisation capacity of Ranpirnase was much slower and became evident only after 4 h and gradually progressed up to 24 h (Fig. 3C).

Cytotoxicity of recombinant proteins towards tumour cells in vitro Antitumour activity of scFv, diabody, Ranpirnase-scFv, Ranpirnasediabody, Ranpirnase and Cetuximab was tested on four lowpassage HNSCC patient-derived cell lines (HNO97, HNO211, HNO410, HNO210), two commercial HNSCC cell lines (FaDu, CAL27), and the epidermoid carcinoma cell line A431 (Fig. 4A). EGFR-negative tumour cell lines MFC7 and Raji were used for controls (Fig. 4B). Incubation of cells with single agent Ranpirnase resulted in significant cytotoxicity towards both EGFR-positive and EGFR-negative tumour cell lines since its cellular uptake is independent of EGFR expression [31]. ScFv and diabody fragment alone mediated minor to moderate effects on cell viability towards EGFR-positive tumour cells only. Fusion of Ranpirnase to the scFv fragment (Ranpirnase-scFv) augmented the scFv-mediated antitumour effect yet to a smaller extent as Ranpirnase alone. In contrast, Ranpirnase-diabody potentiated cytotoxicity by several orders of magnitude when compared to Ranpirnase-scFv and Ranpirnase alone. Accordingly, Ranpirnasediabody exhibited an up to 2032-fold improved tumour cell killing (for primary patient cell line HNO211) in comparison with Ranpirnase-scFv (Table 2). Overall, Ranpirnase-diabody was cytotoxic at low to sub-nanomolar concentrations in all tested EGFRpositive cell lines, as opposed to Cetuximab which achieved a 50% cell kill in only two of the seven investigated EGFR-positive cell lines (Table 2). Furthermore, and remarkably, cytotoxicity of Ranpirnasediabody was highly specific to EGFR-positive tumour cells since viability of EGFR-negative cells entirely remained unaffected (Fig. 4B). Cytotoxicity of Ranpirnase-diabody well correlated with the EGFR expression level of investigated cell lines, rendering tumour cells with high EGFR expression most susceptible to the immunoRNase (Table 2).

Mode of action of Ranpirnase-diabody

Fig. 3. Internalisation of immunoRNases, Ranpirnase and Cetuximab. (A) Individual confocal sections of A431 cells incubated with Cetuximab (top row), RanpirnasescFv (middle row) and Ranpirnase-diabody (bottom row) for various times. (B) Quantification of uptake of Ranpirnase-scFv, Ranpirnase-diabody and Cetuximab normalised to the latest time point examined (means ± SE). (C) Individual confocal sections and quantification of Ranpirnase uptake into A431 cells (means ± SE). Scale bar is 10 μm.

Cetuximab were rapidly internalised into the tumour cell and uptake began to saturate after 1–2 h (Fig. 3A,B). Interestingly, both immunoRNases were more rapidly internalised than Cetuximab. The fastest uptake was observed for Ranpirnase-diabody reaching ~50% of

Major factors of Ranpirnase-mediated cell death are inhibition of protein synthesis through tRNA degradation and triggering of apoptotic pathways via activation of caspases [18]. A dominant mechanism for the antitumour activity of Cetuximab is mediated by the induction of apoptosis through disruption of the EGFR signalling pathway and G1 phase cell cycle arrest [32]. We therefore further analysed the Ranpirnase-diabody fusion protein for apoptosis induction and EGFR downstream signalling alterations. We determined caspases 3 and 7 activity in HNO211 and FaDu being the most and the least susceptible cell lines towards Ranpirnase-diabody treatment, respectively (Fig. 5). Ranpirnase alone upregulated caspase 3/7 activity about 3.1-fold in HNO211 and 1.6fold in FaDu cells, whereas Cetuximab showed only a marginal effect on caspase 3/7 activation in both cell lines. Caspase activation by Ranpirnase-diabody was markedly increased when compared with Ranpirnase-scFv and was similar to that observed for Ranpirnase alone. Ranpirnase and Ranpirnase-diabody induced relatively weak caspase activation in FaDu cells, which is in agreement with the observed lower cytotoxic activity of both the RNase and the immunoRNase towards these cells (Table 2). Effects on EGFR downstream signalling were investigated by quantification of phosphoprotein (and total protein) of key signalling pathways. Observed effects are shown in Fig. 6, focussing on differences between Cetuximab and Ranpirnase-diabody. Raw data of measured ratios (phosphoprotein/total protein) are given in Supplementary Table S1.

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Fig. 4. Antitumour activity of recombinant proteins in vitro. EGFR-positive (A) and EGFR-negative (B) tumour cell lines were incubated with different concentrations of scFv, diabody, Cetuximab, Ranpirnase-scFv, Ranpirnase-diabody or Ranpirnase for 72 h. Cell viability is expressed relative to buffer-treated control cells. Data depict mean values ± SE from one representative experiment performed in triplicates.

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Table 2 Antitumour activity of immunoRNases towards investigated tumour cell lines in comparison to respective antibodies and Ranpirnase. IC50 values are indicated as mean ± SE and are derived from at least two independent experiments each performed in triplicates. IC50 (nM)

scFv Diabody Cetuximab Ranpirnase-scFv Ranpirnase-diabody Ranpirnase EGFR Expression*

A431

HNO211

HNO97

CAL27

HNO410

HNO210

FaDu

MCF7

Raji

>3618 >4064 >4300 >3618 3.4 ± 0.5 107 ± 29 +++

>3618 >4064 535 ± 162 1423 ± 712 0.7 ± 0.3 4±1 +++

>3618 847 40 ± 23 1262 ± 59 15 ± 3 255 ± 38 ++

>4036 >4064 >4300 1423 115 ± 1 123 ++

>3618 >4064 >4300 2524 ± 168 92 ± 1 119 ± 48 +

>4036 >4064 >4300 2073 115 171 ± 4 +

>4036 >4064 >4300 >3618 371 ± 91 281 ± 183 +

>3618 >4064 >4300 >3636 >636 1351 ± 149 −

>4036 >4064 >4300 >3636 >636 698 ± 134 −

* Relative EGFR cell surface expression was analysed by flow cytometry.

In vivo activity of Ranpirnase-diabody To assess tumour and organ distribution of Ranpirnase-diabody in comparison with unfused RNase, tissues of A431 tumourbearing mice were dissected 24 h and 48 h after intravenous administration of 131I-labelled proteins. Biodistribution results demonstrated a specific tumour uptake of Ranpirnase-diabody at 24 h

(1.7% ID/g) and 48 h (0.6% ID/g), corresponding to tumour-toblood ratios of 7.2 and 10.8, respectively (Supplementary Fig. S1A,B). Tumour uptake of Ranpirnase-diabody was EGFR-mediated since no accumulation of radioactivity in this tissue was observed for a CD22-specific Ranpirnase-diabody control fusion protein. For Ranpirnase, most radioactivity accumulated in the kidneys, pointing to rapid elimination of the RNase from the blood circulation. As a consequence, Ranpirnase did not significantly localise to tumour cells, as almost no radioactivity was detected in tumour tissue. The capability of Ranpirnase-diabody to specifically infiltrate tumour tissue was further investigated by scintigraphic imaging (Fig. 7A). In accordance with the biodistribution results, Ranpirnase-diabody revealed a highly specific uptake in the tumour showing maximal uptake values at 24 h. Over the course of the examination period, Ranpirnase-diabody was efficiently cleared from the body. Due to its specific binding, a considerable amount of the immunoRNase was retained in the tumour. At 72 h post injection, scintigraphy revealed an almost exclusive accumulation of Ranpirnase-diabody in tumour tissue, highlighting the excellent tumour specificity of the dimeric immunoRNase. Residual radioactivity found in the thyroid can be attributed to radioiodine cleaved from the protein [33]. To explore the therapeutic efficacy of Ranpirnase-diabody in vivo, nude mice bearing A431 xenograft tumours received 60 μg Ranpirnase-diabody by intraperitoneal injection for five consecutive days, resulting in a total cumulative dose (TCD) of 300 μg per mouse. Control mice received either PBS, 40 μg diabody (TCD = 200 μg) or 20 μg Ranpirnase (TCD = 100 μg). As shown in Fig. 7, mice did not benefit from the treatment with either diabody or Ranpirnase alone and only a small tumour growth delay was observed. In contrast, Ranpirnase-diabody significantly (p < 0.0001) inhibited tumour growth that translated into statistically significant (p = 0.0004) increased overall survival of the mice when compared to the buffer-treated control group (Fig. 7B,C). Most notably, treatment with Ranpirnase-diabody was well tolerated and only induced a moderate weight loss (approximately 14% of initial mouse body weight) that was reversible after cessation of treatment (data not shown). Discussion

Fig. 5. Caspase 3/7 activity. HNO211 and FaDu cells were treated with 100 nM of either scFv, diabody, Cetuximab, Ranpirnase-scFv, Ranpirnase-diabody or Ranpirnase for 24 h. Caspase 3/7 activity is expressed relative to buffer control. Data depict mean values ± SE from triplicates.

To target EGFR-expressing tumour cells we have generated two different immunoRNases by fusion of Ranpirnase to either an anti-EGFR scFv or an anti-EGFR diabody fragment. The dimeric immunoRNase, Ranpirnase-diabody, exhibited improved tumour cell binding, markedly enhanced ribonucleolytic activity and was by several orders of magnitude more toxic to tumour cells than the monomeric immunoRNase Ranpirnase-scFv. Bivalent anti-EGFR antibody formats have been reported to be more effective than employing monovalent antibody fragments for triggering receptor dimerisation [34,35]. Because dimerisation seems

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Fig. 6. EGFR signalling pathways in cell line HNO211. Cells were treated with 100 nM of either Cetuximab, Ranpirnase-diabody or Ranpirnase for 24 h. Effects on EGFR signalling were quantified (ratio of phosphoprotein to total protein) and compared to PBS-treated control. Lines only indicate association between proteins (undirected). Red indicates decreased activation (≤25%), yellow indicates indifferent activation and green indicates increased activation (≥25%) compared to control, respectively.

to enable efficient internalisation of EGFR [36,37], EGFR-targeting with bivalent antibody fragments might provide a more beneficial approach for delivering cytotoxic payloads into the cytosol of EGFR-expressing tumour cells. In the present paper we show that the superior cytotoxicity of Ranpirnase-diabody cannot be attributed to a more efficient internalisation as both the monovalent and the bivalent immunoRNase were very rapidly internalised. In contrast, internalisation of Ranpirnase alone into EGFR-positive tumour cells was relatively slow and only reached its plateau after 24 h. Since time-dependent internalisation of both immunoRNases was even more pronounced than for Cetuximab, we cannot exclude that the combination of EGFR-binding and the net positive charge of Ranpirnase caused a more efficient internalisation. Interestingly, we found that signalling pathway alterations mediated by Cetuximab and Ranpirnase-diabody were profoundly different on the SMAD2 and mTOR axis. While both treatments showed efficient downregulation of EGFR downstream signalling, the main difference between Cetuximab and Ranpirnase-diabody affects differential mTOR signalling. This effect could explain observed differences in the cytotoxicity assays because additional inhibition of the mTOR pathway in the patient cell line HNO211 may have resulted in maximised growth inhibition or cell death induction [38]. Ranpirnase alone showed only slight effects on signalling pathways, highlighting the complementary effects of Ranpirnase fused to the diabody fragment.

Simultaneous delivery of two effector RNase moieties by generating a dimeric immunoRNase resulted in a molecule with highly promising antitumour activity. These results are also consistent with our previous findings that dimerisation of monomeric RNase fusion proteins may significantly increase antitumoural potency of ribonuclease-based fusion proteins [21,22]. The dose-limiting toxicity of single agent Ranpirnase is caused by damage of proximal kidney tubular cells by highly unspecific uptake in this tissue [20,39,40]. Restricting activity of the RNase to only cancerous tissue thus is of fundamental importance for developing a future clinical Ranpirnase-based product. In the present study we showed that linkage of Ranpirnase to a bivalent antiEGFR antibody fragment resulted in selective and specific cytotoxicity only towards EGFR-positive tumour cells whereas Ranpirnase alone mediated cytotoxicity towards both EGFR-negative and EGFRpositive cells. Consequently, treatment of tumour-xenografted mice with the dimeric immunoRNase did not only result in specific tumour uptake as shown by biodistribution analysis and scintigraphic imaging but translated into statistically significant overall survival. In addition, antibody-targeted delivery of Ranpirnase is capable to increase the therapeutic window of the RNase by several orders of magnitude. We have in the present study administered Ranpirnase-diabody intraperitoneally at 67 μg per nude mouse (3.7 mg/kg) for six consecutive days without experiencing severe dose-related complications or death (data not shown). In

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Fig. 7. Activity of Ranpirnase-diabody in vivo. (A) Planar scintigraphic images of an A431 tumour-bearing nude mouse taken 24, 48 and 72 h after injection of 125I-labelled Ranpirnase-diabody. (B) Tumour growth of A431 xenograft-bearing nude mice treated with Ranpirnase-diabody, Ranpirnase, diabody or buffer control at indicated time points (means ± SE). (C) Survival analysis of A431 xenograft-bearing nude mice treated with Ranpirnase-diabody, Ranpirnase, diabody or buffer control.

comparison, the maximum-tolerated dose (MTD) of single agent Ranpirnase intravenously administered to patients with solid tumours on a weekly schedule has been reported to be 960 μg/m2 [20]. The abrogation of non-EGFR-mediated cell entry of Ranpirnase in the fusion protein configuration can be considered as the major factor for increasing the therapeutic window of Ranpirnase and provides a most important prerequisite for further development of this compound into a clinically employable drug. For targeting solid tumours in vivo, pharmacokinetics of antibody constructs need to be considered. Although mAbs possess long serum half-lives that may be favourable for tumour interaction, tumour penetration is limited by their large size (~150 kDa) and mostly restricted to tumour cells in close proximity to blood vessels [41]. Smaller-sized antibody fragments such as scFvs show superior tumour penetration capacity when compared to IgG-formatted mAbs [41,42]. However, scFvs are rapidly cleared from the blood due to their molecular weight well below the glomerular filtration threshold of ~60 kDa [43]. Molecules of intermediate size (60–100 kDa) show decreased systemic

clearance yet are small enough to penetrate tumour tissue efficiently and therefore provide an ideal compromise between tumour penetration and serum half-life [44]. In addition, bivalency results in improved tumour retention when compared to monovalent antibody counterparts [45,46]. The bivalent Ranpirnase-diabody construct with a molecular weight of about 81 kDa can therefore be expected to possess most favourable pharmacokinetic properties as suggested for antibodybased therapeutics [44,47]. Based on its selective antitumour efficacy on EGFR-positive target cells, its expected favourable pharmacokinetics and ease of production in mammalian cell systems, we believe that further development of anti-EGFR Ranpirnase-diabody into a clinical drug is promising. Acknowledgments We thank Karin Leotta, Stephanie Haase, Tina Lerchl, Evelyn Exner and Raphael Bleiler for excellent technical assistance. This work was in part supported by Deutsche Krebshilfe (DKH 108813) and a re-

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